The MAPs of all aged groups, SE-Aged, IM-Aged, and ET-Aged, were significantly higher than that of the SE-Young group. The regional cerebral blood flow (rCBF) in the SE-Aged and IM-Aged was significantly decreased as compared to SE-Young groups. However, rCBF of ET-Aged group was significantly higher than that in the IM-Aged group (P < 0.05). Moreover, the percentage of capillary vascularity (%CV) and the levels of VEGF and eNOS in the ET-Aged group were significantly increased compared to the IM-Aged group (P < 0.05). These results imply that exercise training could improve age-induced microvascular changes and hypoperfusion closely associated with the upregulation of VEGF and eNOS.This study aimed to investigate the effect of exercise training on age-induced microvascular alterations in the brain. Additionally, the association with the protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was also assessed. Male Wistar rats were divided into four groups: sedentary-young (SE-Young,  According to a United Nations report , the worOxidative stress in a physiological setting can be defined as an excessive bioavailability of reactive oxygen species (ROS), which is the net result of an imbalance between the production and destruction of ROS (with the latter being influenced by antioxidant defenses). The “oxidative stress theory” of aging is a prevalent theory that proposes that a progressive and irreversible accumulation of oxidative damage caused by ROS impacts critical aspects of the aging process and contributes to impaired physiological function, an increased incidence of disease and a reduction in life span . ROS areSeveral studies have shown that exercise prevents vascular dysfunction, which is associated with a reduction in oxidative stress , 6. In aAngiogenesis induced by exercise has been reported for its association with an increase in the expression of angiogenic factors. Lloyd et al.  reportedn = 5), sedentary-aged (aged 23-24 months) , immersed-aged , and exercise trained-aged  rats. This study was approved by the Ethics Committee on the Care and Use of Laboratory Animals of the Faculty of Medicine Chulalongkorn University. The present study was conducted in accordance with the guidelines for laboratory animals established by the National Research Council of Thailand (1999).Male Wistar rats were divided into four groups: sedentary-young (aged 4–6 months)  and sedentary-aged (aged 23-24 months). These sedentary rats were subjected to the same swimming-room environment as the exercise trained-aged animals, except they remained in their cages.In this experiment, the age-induced microvascular alterations will be demonstrated by the statistical analysis between sedentary-young (aged 4–6 months)  and the exercise trained-aged  rats. To minimize the possible stress effects associated with cold or hot water exposure, therefore, the IM-aged group was performed according to the modified methods of Iemitsu et al. and Eksakulkla et al. [The effects of exercise training in aging group will be a statistical comparison between immersed-aged . Another catheter was placed in the left femoral vein for the injection of a fluorescent tracer, supplemental anesthesia, and the replacement of fluids.On the day of the experiment, the body weights of the rats, which were fasted overnight, were measured, and the rats were then anesthetized with pentobarbital sodium . Their body temperature was maintained at 37°C with a homoeothermic blanket. The rats were tracheotomies and mechanically ventilated with room air containing supplemental oxygen using an animal ventilator . A catheter was placed into the right femoral artery to obtain a blood sample for measurement of blood gases with a portable blood gas analyzer . The arterial blood gases were measured before and after the experiment, and the blood gas values were recorded and remained stable within normal ranges .Each rat was placed in a homemade stereotaxic apparatus. The animal's head was fixed in the stereotaxic frame, and the skull was exposed via a midline incision. Using a dissecting microscope  and a low-speed drill , a circular cranial window of 3 mm in diameter was created over the left parietal cortex . To prevent the overheating of the cerebral cortex during drilling, the skull was cooled by the periodic application of room temperature saline. The dura mater was carefully removed using a pair of microscissors . A custom-made stainless metal ring (8 mm in diameter) was then sealed over the cranial window using dental cement . The cerebral cortex was suffused with artificial cerebral spinal fluid . The arterial blood gases were sampled before and after the direct imaging of the cerebral microvascular network. To visualize the microvessels, 0.2 mL fluorescein isothiocyanate-dextran (FITC)  was injected intravenously. The microvascular network was imaged using a laser scanning confocal microscopy system . This system included an upright fluorescence microscope equipped with an epi-illuminator for fluorescence, which was provided by a high-pressure mercury lamp  using an appropriate filter for FITC. The light source for the confocal microscope was an argon-ion laser whose wavelength was 488 nm. The cranial window was explored with a 10x objective lens . The cerebrovascular network was recorded using a computer-based frame grabber  with a controlled gain, offset, and exposure time. The microvascular network was visualized and recorded within 5 minutes shown in . The colAfter imaging, a cannula was inserted into the apex of the left ventricle to allow for the perfusion of the brain with 200 mL ice-cold phosphate buffer saline (PBS) containing heparin (25 UI/mL). The brain was then removed and stored at −80°C until the cerebral microvessels were subsequently isolated. The isolated brain microvessels were used for the determination of the VEGF, Flk-1, and eNOS levels by an immunoassay technique.The regional cerebral blood flow (rCBF) was measured by laser Doppler perfusion monitoring with a laser Doppler probe  interfaced to a laptop equipped with the PeriSoft data-acquisition software . Laser Doppler flowmetry is a noninvasive method of determining tissue perfusion and is becoming a widely used technique. Its underlying principle is that the laser light backscattered from the tissue is spectrally broadened by the Doppler shifts produced by moving red blood cells. For each measurement, the laser Doppler probe was placed perpendicular to the cortical surface, and placement over any areas with large vessels was avoided. The rCBF data for each animal was obtained from three cortical regions and is expressed in arbitrary “perfusion units.”μm rectangular region of interest (ROI). Five ROI were selected for each image, and each ROI was oriented to include microvessels (<10 μm in diameter)  . All imaiameter) .The software calculated the percentage of capillary vascularity using the following equation:The determination of capillary vascularity was performed using 5 ROIs/image, 3 images/animal, and 5–8 animals/group.Brain microvessels were isolated according to the method of Yamakawa et al. . The braAt the end of the experiments, whole blood was collected from the abdominal aorta for the determination of the malondialdehyde (MDA) level (a common oxidative stress marker) using a commercial assay kit .μL of ice-cold RIPA buffer containing a protease inhibitor cocktail . The homogenate was centrifuged at 1,000 ×g for 5 minutes at 4°C. The supernatant, as the postnuclear fraction, was aliquot and stored at −80°C. The protein concentration of the postnuclear supernatant was determined using a bicinchoninic acid (BCA) assay kit . The supernatant aliquots were used for the quantification of the VEGF, Flk-1, and eNOS protein levels with commercial immunoassay kits .Isolated brain microvessels were homogenized in 500 The VEGF levels were quantified using a sandwich enzyme immunoassay technique and an ELISA kit . The standard solution or the samples were added to a 96-well plate that was precoated with a polyclonal antibody specific for VEGF. The samples were incubated for 2 hours, and after washing the plate, a substrate solution was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. The intensity of the color was proportional to the amount of VEGF bound in the initial step. The sample values were then compared to the standard curve.Flk-1 expression was measured using a solid-phase sandwich ELISA kit . The standards and samples were pipetted into the wells, and any Flk-1 present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for Flk-1 was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. The intensity of the color measured was proportional to the amount of Flk-1 bound in the initial step. The sample values were then compared to the standard curve.The eNOS protein levels were quantitated using a commercially available solid-phase sandwich ELISA kit  following the manufacturer's instructions. This assay employed a quantitative enzyme immunoassay technique in which a monoclonal antibody specific for eNOS was precoated onto a microplate. The standards and samples were pipetted into the wells, and any eNOS present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for eNOS was added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells, and color developed in proportion to the amount of eNOS bound in the initial step. The color development was stopped after 10° min, and the intensity of the color was measured. The eNOS concentration of each sample was then calculated from the standard curve. t-test for unpaired values was used. Differences were statistically significant if the statistical probability  was less than 0.05. The data were analyzed using SPSS 16.0 for Windows .The results are expressed as the mean ± standard error of mean (SEM). Any significant differences between groups were determined using the one-way analysis of variance (one-way ANOVA), and differences between pairs of mean values were evaluated by the least significant difference (LSD) test. To evaluate the difference in between the immersed-aged and exercise trained-aged groups, Student'sP < 0.05).The body weights and mean arterial blood pressures (MAPs) of the rats in the sedentary-young, sedentary-aged, immersed-aged, and trained-aged groups are summarized in P < 0.05). However, the ET-Aged group had a significantly reduced MAP compared to the IM-Aged group (P < 0.05).The MAPs of the SE-Aged (130.00 ± 5.00 mmHg), IM-Aged (130.39 ± 2.69 mmHg), and ET-Aged (115.79 ± 4.30 mmHg) groups were significantly higher than that of the SE-Young group  in the SE-Aged (134.99 ± 14.74 PU), IM-Aged (131.09 ± 17.53 PU), and ET-Aged (255.83 ± 13.17 PU) groups compared to the SE-Young group (334.27 ± 35.00 PU) (P < 0.05).The rCBF was significantly diminished (5.00 PU) . HoweverP < 0.05) compared with the SE-Young group (39.25 ± 2.18%). The capillary vascularity of the ET-Aged group (29.81 ± 1.64%) was significantly greater than that of the IM-Aged group (16.46 ± 1.59%) (P < 0.05).The effect of exercise training on brain capillary vascularity in aging rats was illustrated in Images of the microvascular networks for the SE-Young, SE-Aged, IM-Aged, and ET-Aged groups were visualized by FITC-dextran under a laser-scanning fluorescence confocal microscope with a 10x objective lens . In the P < 0.05) in the SE-Aged (20.51 ± 1.75 pg/mg protein) and IM-Aged (19.07 ± 1.27 pg/mg protein) groups compared with the SE-Young group (32.27 ± 0.93 pg/mg protein) (P < 0.05) in the level of VEGF in the ET-Aged (28.35 ± 1.53 pg/mg protein) group when compared with the IM-Aged group (19.07 ± 1.27 pg/mg protein). Additionally, there was no significant difference in the VEGF levels between the SE-Young and ET-Aged groups (P = 0.075).The VEGF level was significantly lower (protein) . Swim trP < 0.05) than that in the SE-Young group (0.076 ± 0.021 pg/mg protein) . However, the level of Flk-1 in the ET-Aged group (0.064 ± 0.011 pg/mg protein) was showing more significant increase than that in the IM-Aged group (0.014 ± 0.003 pg/mg protein) .The level of Flk-1 in the IM-Aged group (0.014 ± 0.003 pg/mg protein) was significantly lower (protein) . AlthougP < 0.05) eNOS levels  compared to the SE-Young group (994.39 ± 88.49 pg/mg protein) (P < 0.05) eNOS level in the ET-Aged group (926.75 ± 65.08 pg/mg protein) compared with the IM-Aged group (459.94 ± 98.02 pg/mg protein). However, the level of eNOS in the ET-Aged group was not significantly different (P = 0.5480) from that in the SE-Young group.Both the SE-Aged and IM-Aged groups had significantly lower (protein) . RegularR2 = 0.7958. The linear regression equation was expressed as y = −1.9368x + 40.999.The data on the capillary vascularity and MDA level in the SE-Young, SE-Aged, IM-Aged, and ET-Aged groups were collected and plotted in  in vivo animal study, the protective effects of moderate exercise training on brain microvessels against age-induced microvascular changes were revealed. The deterioration of capillary vascularity and regional blood perfusion was found in the aging brain. Moreover, these results indicated that a moderate exercise training program could attenuate the decreases in brain capillary vascularity and blood perfusion and could significantly reduce age-induced oxidative stress. The upregulation of VEGF and eNOS, which are two key angiogenic proteins, may play a role in the protective effect of exercise training in the amelioration of brain microvascular perfusion during aging.In the presentThe present study demonstrated the alterations in physiological characteristics induced by aging, including changes in body weight, MAP, and oxidative stress . The greThe present data demonstrated that hypertension occurred in the SE-Aged and IM-Aged groups. Multiple mechanisms have been reported for the pathogenesis of hypertension in the elderly, including endothelial dysfunction (loss of vasomotor regulation), the elevation of oxidative stress, inflammation, cellular apoptosis, and increased concentrations of active metabolites (increased myogenic constriction) . InteresThe present data demonstrated that the plasma MDA levels were significantly higher in both the IM-Aged and SE-Aged groups compared to the SE-Young group. In addition, swim training of moderate intensity also produced a lower plasma MDA level in the ET-Aged group compared to the SE-Aged group. Based on the free radical theory of aging, various oxygen species oxidize lipids, resulting in a diverse array of peroxide products . AlthougThe present study demonstrates that an 8-week swim training protocol results in a reduction of body weight and resting MAP as well as attenuated oxidative stress in aging rat model. We therefore conclude that adequate endurance training can be achieved using our swimming exercise program. in situ investigation of cerebral microcirculation through the cranial window. The current method is based on the combined use of fluorescent intravascular tracers and laser-scanning confocal fluorescence microscopy and was developed for the real-time in vivo study of cerebral microcirculation in rats. The main advantage of laser-scanning confocal fluorescence microscopy is that it makes possible the high-quality visualization of the microvascular network from the surface through to the intraparenchyma at a depth of 100–200 μm. For short periods of in situ exploration, the brain tissue was not damaged by the laser illumination, and the normal physiological status was maintained .The present study describes our directA reduction in cerebral blood flow has been found to occur with advancing age. The present study confirmed this phenomenon by demonstrating a markedly lower regional cerebral blood flow in both the SE-Aged and IM-Aged groups . The impAge-related brain microvascular rarefaction has the potential to result in inadequate blood flow to the brain. The mechanism for age-related changes in the brain microvasculature appears to correlate, at least in part, with a decline in growth factors that mediate angiogenesis. Vascular endothelial growth factor (VEGF) is a likely regulator of angiogenesis in the peripheral and central vascular systems. In the presence of VEGF, angiogenesis occurs, but in the absence of VEGF, the capillaries undergo apoptotic regression . There aIn vitro and in vivo experiments have demonstrated that eNOS enhances endothelial cell migration, proliferation, and differentiation [ntiation –39. It hntiation . The stuntiation . The preThe angiogenic action of VEGF is mainly mediated by VEGF receptor 2 (KDR/Flk-1), which is primarily expressed by endothelial cells. The activation of the tyrosine kinase receptor Flk-1 in angiogenesis has been shown to be associated with the activation of eNOS in endothelial cells . In aginA large cross-sectional study provided evidence in humans that the resting blood flow velocity in the middle cerebral artery is elevated by habitual exercise across different ages . In the Nitric oxide, which is produced by endothelial cells, is a regulating factor involved in CBF regulation . It was  in vitro [ in vivo data [ in vivo involves the activation of NOS and the generation of NO [ in vitro [ in vivo study determined that increased capillary shear stress induces angiogenesis [Although the mechanism by which regular physical activity induces beneficial effects in the cerebral vasculature has not been elucidated, it is possible that the repeated exposure to increases in blood flow and shear stress in specific regions of the brain during exercise play a role . The prein vitro  and in vivo data . Vasculaivo data . For theon of NO . Recentlon of NO . Fluid sin vitro . Moreoveogenesis .It has been well established that mitochondrial DNA accumulates mutations with aging . A decreOur findings indicate that the effective mechanisms of exercise training on age-induced brain microvascular changes involved the upregulation of VEGF and eNOS expression in association with changes in the oxidant-antioxidant balance.


Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis. Brucella. This genus includes several species among which B. abortus preferentially infects cattle, B. suis swine and wild-life and B. melitensis goats and sheep. These three species are zoonotic and cause a grave and debilitating disease in humans. Sheep can also be infected by B. ovis, a non-zoonotic species. B. ovis brucellosis is characterized by a decreased fertility in rams, occasional abortions and a rise in perinatal mortality ]. Th]. ThBrucucellae .Brucella species ], an], anBruclipid A . These esponse .B. melitensis or B. ovis can be controlled by vaccination with the attenuated strain B. melitensis Rev 1 and, in fact, this is the only effective way to control sheep brucellosis in areas with a high or moderate prevalence of the disease ]. A]. AB. meesearch , recombesearch  or DNA esearch . Howeveesearch ], w], wB. meB. ovis virulence and to develop B. ovis-specific vaccine candidates that could overcome the above-summarized drawbacks. To this end, we built on previous genetic analyses that have shown that at least three glycosyltransferases  are necessary for the complete assembly of the core oligosaccharide of B. abortus and B. melitensis ,17],[17]B. o,[17]B. oitensis .B. ovis PA is a virulent strain isolated from a naturally infected ram that has been extensively used as a challenge for the evaluation of B. ovis vaccines in rams and mice. B. ovis strains were cultured on tryptic soy agar  or in tryptic soy broth  supplemented with 0.5% yeast extract  or on Blood Agar Base No. 2 , all supplemented with 5% porcine or calf serum . Incubations were performed at 37 °C in a 10% CO2 atmosphere, and liquid cultures were shaken at low intensity. E. coli was grown in Luria-Bertani broth . Nalidixic acid , Kanamycin , Gentamicin  or sucrose (5% w/v)  were used when required.The bacterial strains and plasmids used in this work are listed in Table Plasmid and genomic DNA were isolated with Qiaprep Miniprep  and Ultraclean Microbial DNA Isolation Kit , respectively. When needed, DNA was also purified from agarose gels using a Qiack Gel extraction kit (Qiagen). DNA sequencing was performed by the dideoxy method at the Sequencing Unit of Centro de Investigación Médica Aplicada , and primers were synthesized by Sigma-Aldrich Ltd. Searches for DNA and protein homologies were carried out using the Kyoto Encyclopedia of Genes and Genomes , EMBL-EB. ovis PA as a DNA template. Primers were designed based on the sequence of B. ovis ATCC 25840 . They are listed in Table wadC (BOV_1453), we first generated two PCR fragments: the 5′end of the gene BOV_1453 was amplified with primers wadC-F1 and wadC-R2 obtaining a 473-bp fragment including codons 1 to 16 of the wadC ORF, as well as 424-bp upstream of the wadC start codon; whereas the 3′end was amplified with primers wadC-F3 and wadC-R4 obtaining a 481-bp fragment including codons 307 to 355 of the wadC ORF and 315-bp downstream of the wadC stop codon. Both fragments were ligated by overlapping PCR using primers wadC-F1 and wadC-R4. The resulting fragments containing the wadC deletion allele, was cloned into pCR2.1 vector to generate plasmids pYRI-14  was selected by Nal and Km resistance, and the second  by Nal and sucrose resistance and Km sensitivity. The resulting colonies were screened by PCR with primers wadC-F1 and wadC-R4, which amplify a fragment of 934-bp in the mutant strain and a fragment of 1804-bp in the parental strain. An additional PCR was carried out to exclude the presence of the complete gene. The amplification was done with primers wadC-F1 and wadC-R5, which include a fragment from the deleted region of the gene. While strains carrying the complete gene amplified a 533-bp fragment, the mutant strain was unable to amplify the fragment  was obtained in a similar way. Briefly, primers wadA-F1 and wadA-R2 were used to amplify a 453-bp fragment including codon 1 to 57 of the 5′end of wadA (BOV_0614), as well as 284-bp upstream of the wadA start codon; and primers wadA-F3 and wadA-R4 were used to amplify a 460-bp fragment including codons 680 to 704 of the wadA ORF and 386-bp downstream of the wadA stop codon. Both fragments were ligated by overlapping PCR using primers wadA-F1 and wadA-R4, the resulting fragment was cloned into pCR2.1, sequenced and subcloned into pJQK. The resulting mutator plasmid  as described before ]. E]. E32]].]. E]. E3600 evolution of a bacterial suspension in TSB-YE-S, over 48 h of static incubation at room temperature, starting from initial readings of 0.8 (100% OD600) in TSB-YE-S ]), ]), B. ab1_0351, ) and waB. ovis LPS using a SDS-proteinase K protocol ]. I]. IB. ab]. I]. IB198 LPS , is in B. abortus and B. melitensis mutants is altered, the O-polysaccharide remains in place . This evidence shows that glycosyltransferases WadB and WadC are responsible for the synthesis of the glucosamine rich oligosaccharide stemming from Kdo II  is essential for survival in the mouse model. Even though the exact mechanism remains to be elucidated, the proposed core defect is likely to facilitate the recognition of B. ovis LPS by innate immunity. Indeed, this hypothesis is supported by the above-summarized results obtained with the homologous B. abortus and B. melitensis mutants and with the fact that, rather than the O-polysaccharide, it is the lipid A-core overall structure that is recognized by multiple innate immunity systems ]. N]. NB. abn power ].]. N]. NBBrucella effectively evade a prompt detection by innate immunity ) that covers the use of PS-L carried out the experiments and participated in the design of the study and draft of the manuscript. YG-R, AZ, MI, RC-A and NV collaborated with the design and construction of the mutants. MZ and AC helped with the immunochemical studies and the characterization of the LPS. M-JG, AZ-B and BSR participated in mice in vivo studies. IL-G and IM conceived the study, participated in its design, coordinated the work and helped to draft the manuscript. All authors read and approved the final manuscript.Confirmation of the construction of the mutants. A: schematic representation of the ORF indicating in grey colour the deleted region and the primers used to check the strains. B: For each gene, two confirmation PCR assays were carried out with specific primers. Primers F1-R4 flank the ORF and show different sizes between the mutant and the wild type gene; and primers F1-R5 cover from upstream of the ORF to the deleted region, so that only strains carrying the complete ORF will be amplified and the specific amplicon will be shown. (1) 1 kb plus ladder marker; (2) B. ovis PA; (3) B. ovis deleted mutant; (4) B. ovis sibling revertant strain.Click here for file


Schistosoma mansoni infection, but suffers from low sensitivity when infections intensities are moderate to low. Thus, misdiagnosed individuals remain untreated and contribute to the disease transmission, thereby forestalling public health efforts to move from a modality of disease control to one of elimination. As an alternative, the urine-based diagnosis of schistosomiasis mansoni via the circulating cathodic antigen immuno-chromatographic test (CCA-ICT) has been extensively evaluated in Africa with the conclusion that it may replace the KK test in areas where prevalences are moderate or high.The Kato-Katz (KK) stool smear is the standard test for the diagnosis of S. mansoni soluble egg antigen (SEA) and soluble (adult) worm antigen preparation (SWAP). Sensitivity and specificity were influenced by whether the trace score visually adjudicated in the CCA-ICT was characterized as positive or negative for S. mansoni infection. An analysis of a two-graph receiver operating characteristic was performed to change the cutoff point. When the trace score was interpreted as a positive rather than as a negative result, the specificity decreased from 97.6% to 78.0% whereas sensitivity increased from 68.7% to 85.4%. A significantly positive correlation between the CCA-ICT scores and egg counts was identified . However, the CCA-ICT misdiagnosed as negative 14.6% of 2KK positive individuals, predominantly those with light infections (fewer than 100 eggs/g feces). Considering 2KK as reference test, the discriminating power of the CCA-ICT (the area under the curve [AUC] = 0.817) was greater than the SEA-ELISA (AUC = 0.744) and SWAP-ELISA (AUC = 0.704).The objective was to measure the performance of the CCA-ICT in a sample study population composed of residents from non-endemic and endemic areas for schistosomiasis mansoni in two municipalities of Minas Gerais state, Brazil. Volunteers (130) were classified into three infection status groups based on duplicate Kato-Katz thick smears from one stool sample (2KK test): 41 negative individuals from non-endemic areas, 41 negative individuals from endemic areas and 48 infected individuals from endemic areas. Infection status was also determined by the CCA-ICT and infection exposure by antibody ELISA (enzyme-linked immunosorbent assay) to i.e., in areas with greater infection prevalence and intensities, the CCA-ICT may be useful as a tool to indicate community-based preventative chemotherapy without individual diagnosis. However, because of the Brazilian Ministry of Health’s recommendation for individual diagnosis in areas where prevalence is less than 15%, i.e., those areas in which infection intensities are likely to be lowest, the CCA-ICT lacks the sensitivity to be used as standalone diagnostic tool.Our data for the performance of the CCA-ICT in the Brazilian communities endemic for schistosomiasis mansoni support those from Africa,  Schistosoma mansoni. However, the test can miss those who have low burdens of infection, i.e., with few eggs in their feces. These misdiagnosed individuals, therefore, do not receive drug treatment and can continue to transmit the parasite into the environment putting the community at risk of infection. As an alternative diagnostic approach, the circulating cathodic antigen immuno-chromatographic test (CCA-ICT) is a simple-to-use handheld device (similar to a pregnancy test) that only needs urine to provide a quick and visual indication of whether one is infected or not. The consensus from studies in Africa is that the CCA-ICT could replace the KK test in those areas where people are more likely to harbor moderate to high worm burdens , but, like the KK test, it can miss those harboring light infection intensities. We evaluated the CCA-ICT performance in urine samples from 130 individuals living in areas non-endemic and endemic for schistosomiasis mansoni within the municipalities of Governador Valadares and Manhuaçu, Minas Gerais state, Brazil. The CCA-ICT performance characteristics, chiefly, sensitivity and specificity, depended on whether a ‘trace’ visual reading of the test was considered as a positive or negative diagnosis. We noted a positive correlation between the CCA-ICT scores and egg counts. However, the CCA-ICT misdiagnosed as negative about 15% of KK positive individuals, predominantly those with light infections. The CCA-ICT, nonetheless, had better discriminating power than commonly used antibody-based tests. We conclude that the CCA-ICT offers reasonable performance to diagnosis S. mansoni infection. However, in areas where infections intensities are light, the test lacks the sensitivity to be used as standalone diagnostic tool.Detecting parasite eggs in stool by the Kato-Katz (KK) stool smear is the standard diagnostic test for infection with the flatworm parasite,  Schistosoma blood fluke that resides in the cardio-vascular system. Official estimates are that 200 million are infected [Schistosoma japonicum, Schistosoma haematobium and Schistosoma mansoni, the latter is the solely responsible for disease in South America, including Brazil [Schistosomiasis is a chronic and morbid global disease caused by the infected ,2 but itinfected ,4 The dig Brazil .http://www.unitingtocombatntds.org/).Treatment and control of schistosomiasis relies solely on the World Health Organization (WHO) recommended drug, praziquantel (PZQ) , 6–10. PS. mansoni and S. japonicum) [As PZQ administration increases, the incidence and prevalence of schistosomiasis are expected to decrease. Of concern, however, is that further progress to drive prevalence and incidence to zero is hindered by the insensitivity of the current ‘gold standard’, diagnostic Kato-Katz (KK) test to detect and facilitate treatment of intestinal schistosomiasis (caused by ponicum) –20. The ponicum) . Howeverponicum)  for commponicum)  stratifiponicum) –28, whicponicum) ,29–33. Cponicum) . Finallyponicum) ,36–37. TImmunodiagnosis is generally more sensitive than examination of stool, particularly in low transmission areas in which infection intensities are light . Typicalhttp://www.rapid-diagnostics.com/), measures a parasite-derived ‘circulating cathodic antigen (CCA)’ in the urine of patients infected with S. mansoni. The recent data (referenced below) suggest that the CCA-ICT performance is good enough to warrant its application as a standalone tool in surveying disease prevalence and risk, and, thus, facilitate the deployment of PZQ therapy at the community level.Over the last several years in sub-Saharan Africa, major efforts, not least the SCORE initiative funded via the Bill and Melinda Gates Foundation  and refeOur report measures the performance of the CCA-ICT in a sample study population comprising residents from non-endemic and endemic areas for schistosomiasis mansoni in Minas Gerais state, Brazil. We compared the performance of the CCA-ICT with the SEA-ELISA and SWAP-ELISA. From the data arising, we interpret the potential utility of the CCA-ICT in the context of how chemotherapy of schistosomiasis is promoted in Brazil.Ethical clearance for this study was obtained from the Ethical Research Committee of Universidade Vale do Rio Doce-UNIVALE (PQ 019/08-11). District health, participants and parents/legal guardians were informed about the purpose and procedures of the study. Parent/legal guardians provided written informed consent for their children to participate. Participation was voluntary and individuals could withdraw at any time and without further obligation. All parasitological results were coded and treated confidentially. Any individual who was determined to be positive for schistosomiasis, soil-transmitted helminthiases or intestinal protozoal infections was treated with the appropriate dose of PZQ, albendazole or metronidazole, respectively, produced and distributed by the Brazilian Ministry of Health .S. mansoni eggs on at least 1 of 2 slides . The 2KK-NEG endemic area individuals had a previous history of at least three negative stool tests for S. mansoni despite being exposed to sources of infection. The 2KK-NEG non-endemic subjects had no previous history of S. mansoni infection, and neither received treatment for schistosomiasis nor had contact with sources of infection. Detailed information for the study groups is presented in The study was conducted from August 2012 to December 2013 in Governador Valadares and Manhuaçu, two municipalities in Minas Gerais state, Brazil . We enroS. mansoni was performed using the 2KK test (41.7 mg of stool per smear) [Ascaris lumbricoides and Trichuris trichiura) and Enterobius vermicularis. Each stool sample was also assessed by the HPJ sedimentation method for helminth eggs and protozoa (Entamoeba sp. and Giardia lamblia) [S. mansoni co-infection with other helminths (only nine out of 130 subjects), the effect of other helminth infections on CCA-ICT performance was not studied further.Diagnosis of r smear) . The intr smear) ,41 was clamblia) . The exaS. mansoni CCA using the CCA-ICT. Urine samples were initially stored at -20°C then prepared as aliquots of 1 ml and frozen at -80°C before use. The CCA-ICT was performed according to the manufacturer’s instructions. Briefly, one drop of urine was added to the sample well of the ICT cassette and allowed to absorb. Then, one drop of buffer (provided with the kit) was added. The test score was read 20 min after adding the buffer. Positive color reactions were scored as trace (very light band), weak (+), medium (++) and strong (+++).A single urine sample from each individual was collected to test for the presence of S. mansoni cercariae of the LE strain. After 45 days, the animals were sacrificed by cervical dislocation and perfused via the hepatic portal system using a 0.85% saline solution containing 50U/l heparin [g for 1 h at 4°C . The supernatant was dialyzed using a cellulose membrane  against 0.9% saline for 48 h at 4°C. The material was then centrifuged at 1,250 g for 15 min at 4°C and the supernatant stored at -20°C. An aliquot was measured for protein content  in order to normalize protein concentration in the SWAP-ELISA used to detect human antibodies (see below).Female Swiss Webster mice (4–6 weeks old) were infected subcutaneously with 100  heparin . Adult wg for 1 h at 4°C. After 48 h of dialysis with a cellulose membrane (Sigma-Aldrich) against a 0.9% saline solution, an aliquot of the supernatant was measured for protein content (Nanodrop) to normalize protein concentration in the SEA-ELISA used to detect human anti-egg antibodies (see below).After perfusion of infected mice, livers were removed to recover parasite eggs. The eggs were homogenized and mechanically ground (Virtiz Precisa) for 40 min in 0.85% saline. The homogenate was centrifuged at 9,500 2O2 Invitrogen T0440, St Louis, USA) were added to each well. The reaction was stopped after 20 min of incubation in the dark by addition of 50 μl/well of 2N sulfuric acid. Absorbance was measured at 450 nm in a microplate reader . Each serum sample was used in duplicate in two separate assays. Average levels of optical density (OD) were determined from the quadruplicate measurements.ELISA tests were performed according to . MicrotiFor the SWAP-ELISA, 1 μg/ml of SWAP was employed. Serum samples were diluted 1:50 and the conjugated secondary antibody was diluted 1:60,000. For the SEA-ELISA, 3 μg/ml of SEA was employed. Serum samples were diluted 1:150 and the conjugated secondary antibody was diluted 1:40,000.We used a judgmental sampling and a non-probability selection approach. Data were analyzed using GraphPad Prism software 5:03, STATA version 13  and SPSS, software version 11.5. Differences were considered significant when α was 0.05. The relationship between the intensity of infection determined by the 2KK test and the CCA-ICT results was examined by the Spearman correlation test. The chi-square test was used to analyze categorical variables. The power of association (the degree of agreement) between the 2KK and CCA-ICT was evaluated using Kappa (k) statistics where k <0 indicates an absence of agreement; k = 0 to 0.2 is poor concordance; k = 0.21 to 0.4 is weak agreement; k = 0.41 to 0.6 is moderate agreement, k = 0.61 to 0.8 is good agreement and k = 0.81 to 1.0 means excellent agreement ,45. The The CCA-ICT results were first categorized based on the adjudicated visual scores as represented in ROC curve analysis indicated that the trace score should be considered negative in order to segregate the “2KK-NEG non-endemic area” from the “2KK-POS endemic area” . Sensitip = 0.0001; p<0.0001) was determined indicating a satisfactory agreement between the tests.As demonstrated above, 31% of “2KK-POS endemic area” individuals were misclassified as uninfected  by the CCA-ICT when ‘trace’ was considered a negative result. Two-graph receiver operating characteristic (TG-ROC) analysis was, therefore, performed to recalculate the cutoff in order to enhance sensitivity. An advantage of the TG-ROC curve approach over the ROC curve is the more direct reading of the value of a cutoff point associated with a specific combination of sensitivity and specificity. The changes in the CCA-ICT performance when the TG-ROC curve cutoff was shifted from ‘trace’ considered as negative to 'trace' considered as positive are shown in p = 0.3499) nor “2KK-NEG non-endemic areas” (P = 0.1000), suggesting that intestinal protozoa infections do not influence the urine CCA-ICT results , NPV (82.1%), specificity 78%) and PPV (82%) of the CCA-ICT  were com for PPV –59,61,70 for PPV ,65,74,80 for PPV ,78–79. H% and PPV for PPV . Our ROCThe exceptions to the Brazilian Ministry of Health’s recommendation of individual diagnosis prior to treatment are in areas where prevalence rates are between 15 and 25% (household co-inhabitants may receive treatment without diagnosis) and above 25% whereby community therapy may be administered to school children and preschoolers without prior diagnosis . In thesS1 Table(PDF)Click here for additional data file.


We examined 1) changes in smoking and vaping behavior and associated cotinine levels and health status among regular smokers who were first-time e-cigarette purchasers and 2) attitudes, intentions, and restrictions regarding e-cigarettes.We conducted a pilot longitudinal study with assessments of the aforementioned factors and salivary cotinine at weeks 0, 4, and 8. Eligibility criteria included being ≥18 years old, smoking ≥25 of the last 30 days, smoking ≥5 cigarettes per day (cpd), smoking regularly ≥1 year, and not having started using e-cigarettes. Of 72 individuals screened, 40 consented, 36 completed the baseline survey, and 83.3% and 72.2% were retained at weeks 4 and 8, respectively.Participants reduced cigarette consumption from baseline to week 4 and 8 (p’s < 0.001); 23.1% reported no cigarette use in the past month at week 8. There was no significant decrease in cotinine from baseline to week 4 or 8 (p’s = ns). At week 8, the majority reported improved health (65.4%), reduced smoker’s cough (57.7%), and improved sense of smell (53.8%) and taste (50.0%). The majority believed that e-cigarettes versus regular cigarettes have fewer health risks (97.2%) and that e-cigarettes have been shown to help smokers quit (80.6%) and reduce cigarette consumption (97.2%). In addition, the majority intended to use e-cigarettes as a complete replacement for regular cigarettes (69.4%) and reported no restriction on e-cigarette use in the home (63.9%) or car (80.6%).Future research is needed to document the long-term impact on smoking behavior and health among cigarette smokers who initiate use of e-cigarettes. Electronic cigarettes (or e-cigarettes) are battery-powered devices that contain liquid that is vaporized with inhalation of the e-cigarette without any combustion or smoke and consist of a combination of propylene glycol, vegetable glycerine, various levels of nicotine, and flavor concentrates . There ii.e., smoking 15 cigarettes per day [cpd]) experimenting with e-cigarettes for 24 weeks (27 retained at follow-up) [A number of studies have demonstrated the efficacy of e-cigarettes in alleviating cravings for cigarettes  5]. Mor. Mor5]. llow-up) . They rellow-up) . A crossllow-up) . This stOnly two randomized controlled trials (RCTs) to date have examined the effects of e-cigarette use on smoking cessation. One study of 657 smokers motivated to quit in New Zealand who were randomized to nicotine e-cigarettes, patches, and placebo e-cigarettes found that, at 6 months, verified abstinence was higher (7.3%) among those using nicotine e-cigarettes versus those using patches (5.8%) and those using placebo e-cigarettes (4.1%); they also found greater reduction in cpd in the e-cigarette group than the patches group . AnotherSources of information regarding e-cigarettes, attitudes about e-cigarettes, and reasons for use have received limited attention in research. Regarding sources of information, one study  found thThere is controversy regarding e-cigarettes and how e-cigarettes should be regulated. Proponents of e-cigarettes indicate that e-cigarettes may result in harm reduction, whereas opponents argue that too little is known about the health impact of e-cigarettes and effectiveness of e-cigarettes in supporting cessation, among other concerns . Given tThe current research protocol was reviewed and approved by the Emory University Institutional Review Board. This study took place in Atlanta, where there were no official policies or restrictions regarding e-cigarette use during this study. Participants were recruited from a community partner, an e-cigarette vendor who has two stores in the metro-Atlanta area and that exclusively sells e-cigarettes that have a cartomizer storing e-cigarette liquid in a reservoir, along with a wide range of liquids in terms of nicotine dose and flavors. Participants were recruited through fliers posted in the store.Eligibility criteria included being ≥18 years old, smoking ≥25 of the last 30 days, smoking ≥5 cpd, smoking regularly for ≥1 year, and not having started using e-cigarettes. Interested participants obtained a study packet from the e-cigarette vendor staff (who volunteered to distribute packets) and then called the research staff while in the e-cigarette store and were screened for eligibility. Eligible participants were instructed to complete the baseline study assessment and mail it to the research office using the pre-addressed stamped envelope. The study packet included two consent forms, one of which they returned completed along with the assessment and one of which they kept for their records.The sample of 36 participants was recruited from July 2013 to September 2013. Of 72 people screened for eligibility, 42 were eligible. Of the 42 eligible, 36 completed and returned the baseline assessment. Retention rates at week 4 and week 8 were 83.3% (n = 30) and 72.2% (n = 26), respectively. Our sample size of completers at weeks 4 and 8 (n = 30 and n = 26) provided us with 80% power to detect an average of 3 cpd and 4 cpd change in cpd from baseline to each follow-up, respectively. There were no significant differences between those retained and those lost to follow-up.Participants completed mail-based, pencil-and-paper survey assessments at baseline, 4 weeks, and 8 weeks, receiving a $75 Amazon gift card for each assessment completed. Each follow-up assessment (conducted at week 4 and week 8) was mailed to participants, and participants received up to six reminder calls and/or text messages to notify them that the assessments were due.Participants reported their age, gender, race/ethnicity, employment status, and education level .At each assessment, participants reported the number of days they smoked in the past 30 days, average cpd, whether they mainly used menthol cigarettes, and brand of cigarettes typically used . They alParticipants reported the level of importance of and confidence in quitting on a 10-point scale . ParticiAt baseline, participants responded to a range of newly developed assessments to examine this emerging behavior for which there are few (if any) standardized behavioral assessments. Participants were asked if they purchased tobacco flavored liquid for their e-cigarettes. We also asked participants, “What strength of e-cigarette liquid did you obtain?” Response options included: “0 mg/ml; 6 mg/ml; 12 mg/ml; 16 mg/ml; 18 mg/ml; 26 mg/ml; or I don’t know  is allowed in some places or at some times” ; or “Smoking [or vaping] is allowed anywhere inside the home . Pa. Pa22]. In order to capture retrospective reports of intentions for e-cigarette use and experiences of initial e-cigarette use, participants were asked a number of open-ended questions at week 4. These questions are listed in Participant characteristics were summarized using descriptive statistics, and paired samples t-tests were conducted to examine differences from baseline to week 4 and to week 8. We also examined the impact of regular cigarette smoking and e-cigarette use in the past seven days on cotinine levels at week 4 and week 8 using ordinary least squares regression. Specifically, we entered average regular cpd and number of times of use of e-cigarettes per the seven-day timeline follow-back assessments of each product. All statistics were conducted using SPSS 21.0, and alpha was set at 0.05. For the open-ended questions, responses were reviewed and categorized by major themes , and representative quotes from responses were selected to qualitatively describe select phenomena and presented in The study sample was an average age of 36.06 (SD = 15.28) years, with 55.6% (n = 22) being female, and 88.9% (n = 32) being White. In addition, 19.4% (n = 7) were unemployed, 58.4% (n = 21) had some college education, 19.5% (n = 7) earned at least a Bachelor’s degree.Baseline measures of smoking characteristics see , indicatRegarding the e-cigarette liquid, 58.3% (n = 21) of participants purchased a tobacco-flavored liquid. Regarding nicotine strength, 11.1% (n = 4) purchased 12 mg/ml, 25.0% (n = 9) purchased 16 mg/ml, 30.6% (n = 11) purchased 18 mg/ml, and the remainder could not remember.There was no significant decrease in cotinine from baseline to week 4 (p = 0.389) or to week 8 (p = 0.387). In the regression examining cotinine levels at week 4, neither average cpd in the past 7 days per the 7-day timeline follow-back  nor the average times using the e-cigarette  predicted cotinine levels (R-squared = 0.052). However, in the regression model examining cotinine levels at week 8, average cpd for the past 7 days predicted cotinine levels , but average times using the e-cigarette did not . In terms of change in health symptoms, at week 8, the majority of participants reported a range of health benefits. There was also no significant difference in average cotinine levels between those who reported 30-day point prevalence abstinence at week 8 versus those who continued to smoke .Most participants  had heard about e-cigarettes from friends and family, while the remainder  heard about them from TV news stories or advertisements see also . In addiMost reported that they intended to use e-cigarettes as a complete replacement for regular cigarettes (69.4%). Fewer participants reported that they were planning to use e-cigarettes where they cannot use regular cigarettes (25.0%) or that they were just experimenting (8.3%) or trying something different  reported no restriction on e-cigarette use. In addition, 61.1% reported that vaping was allowed at work .This study is one of the first to examine the experiences of regular smokers as they initiate use of e-cigarettes. We found that 23.1% reported no cigarette use in the past 30 days at week 8, and the vast majority reported lower cigarette consumption at follow-up. Moreover, participants reduced their average number of times per day using their e-cigarettes from week 4 to 8. These findings add to the body of literature indicating that e-cigarettes may be useful in reducing cigarette consumption among regular smokers who initiate use –[8] [23]It is important to note that there were no significant decreases in cotinine from baseline to week 4 or 8. This is not surprising, given that e-cigarettes contain nicotine and result in escalated cotinine levels –27]. Mo. Mo27]. We also found that the vast majority of participants had heard about e-cigarettes from friends and family. In addition, the majority had not asked their doctor about using e-cigarettes to quit smoking. Of those who had asked their healthcare provider about them, one-third said their doctor recommended that they try it, and the other two-thirds reported that their doctor said that they did not know enough about them. Given the limited empirical evidence of the effectiveness of e-cigarettes in promoting or supporting cessation, more research is needed immediately to inform both consumers and healthcare providers. Moreover, the overwhelming majority of participants believed that e-cigarettes have fewer health risks than regular cigarettes, should be allowed where smoking is not, and have been shown to help smokers quit and to reduce cigarette consumption. Interestingly, only 27.8% believed that the FDA should regulate e-cigarettes. This may be related to relatively little understanding of the role of the FDA in protecting and informing consumers. Alternatively, these consumers may generally reject high levels of regulation and assume that regulation will impact access to e-cigarettes. Further research is needed to understand consumer perceptions of the FDA and its role.Most participants reported that they intended to use e-cigarettes as a complete replacement for regular cigarettes, and a quarter of participants reported that they were planning to use e-cigarettes where they cannot use regular cigarettes or that they were just trying something different. This is consistent with a previous study  that fouFurthermore, participants generally reported positive experiences with e-cigarettes in comparison to regular cigarettes and to other cessation resources. Participants reported believing that e-cigarettes are healthier for the user and those around them, that they satisfy the behavioral component of smoking, and that they address withdrawal symptoms, which aligns with previous findings  29]..29].Regarding restrictions about e-cigarette use in personal spaces and at work, most reported no restriction of use. In addition, most reported that vaping was allowed at work, while fewer reported that smoking was allowed. In contrast, many participants reported restrictions applying to cigarette smoking in home, in the car, and at work. The rates of smoking restrictions are promising; however, additional information is needed to understand the true impact of e-cigarette use among those exposed to secondhand vapor, as prior research indicates that using an e-cigarette indoor may involuntarily expose nonusers to nicotine but not to toxic tobacco-specific combustion products  31]..31].The majority of participants used liquids with nicotine concentrations between 12 mg/ml and 18 mg/ml at all three time points, which is similar to the average of 16 mg/ml found by Etter and colleagues . In addiThis study has some limitations. First, this study was a small pilot study on a small budget that prohibited more elaborate data collection. As such, the small sample size restricts our power. In addition, our weeks 4 and 8 retention rates were 83.3% and 72.2%, respectively. Those lost to attrition may have been more likely to have stopped using the e-cigarette or to have continued smoking regular cigarettes. There may also be bias inherent in recruitment through a local e-cigarette vendor, especially given the variety of e-cigarette products on the market. We also did not assess type of e-cigarette purchased; however, our recruitment site—the e-cigarette vendor— indicated that the majority of first-time purchasers of e-cigarettes at this shop purchase the refillable tank style. We also cannot ensure that participants completed the baseline assessments prior to initiating e-cigarette use. However, we recorded the number of days between initial contact to verify eligibility and receipt of the assessment; all packets were received in the mail within 8 days. In addition, the sample was drawn from individuals living in one southeastern US state, further limiting its generalizability. Furthermore, future research might benefit from a longer follow-up period and additional biomarker assessments . On a related note, exposure to secondhand smoke was not assessed and could not be accounted for in the analyses examining predictors of salivary cotinine.The current study documented significant decreases in cigarette consumption from baseline to week 4 and to week 8 as assessed by all measures. In addition, 23.1% reported no cigarette use in the past 30 days at week 8. Moreover, participants reduced their use of e-cigarettes from weeks 4 to 8. Most participants intended to use the e-cigarette as a total replacement for regular cigarettes and had highly favorable experiences with the e-cigarettes in comparison to regular cigarettes compared to other cessation aids. Finally, we documented very low rates of personal rules restricting where e-cigarettes are used, suggesting that additional research is needed to examine the impact of secondhand vapor on those around e-cigarette users.


P. aeruginosa; however, description of P. aeruginosa co-harbouring two or more carbapenemases is unusual.Carbapenemase genes are one of the most frequent mechanisms reported in carbapenem-resistant P. aeruginosa isolates obtained from a hospital over a 12-year period. A total of 127 isolates of carbapenem-resistant P. aeruginosa recovered from 109 patients feces (four samples), rectal swab (three samples), nasal swab (one sample) and anal abscess (one sample), were evaluated. Minimum inhibitory concentrations of the following antibiotics imipenem, meropenem and polymyxin E were determined by broth microdilution. The molecular profile of isolates was evaluated by pulsed field gel electrophoresis (PFGE). PCR for the following carbapenemase genes blaIMP;blaSPM;blaVIM;blaSIM;blaNDM;blaKPC;blaGES and nucleotide sequencing to confirm the enzyme gene types were performed and compared with the database available on the Internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/).In this study we evaluated the presence of carbapenemase genes and the clonality of 50 and MIC90 were respectively 64 μg/mL and 256 μg/mL to imipenem and 32 μg/mL and 256 μg/mL to meropenem, all isolates except one (MIC = 8 mg/L) were susceptible to polymyxin E. The most frequent carbapenemase genes identified were blaSPM identified in 41 isolates (32%), followed by 10 with blakpc and 5 with blaVIM (3.9%). All belonged to the class SPM-1 and VIM-2. In 2011, one isolate harbouring three carbapenemase genes  that belonged to a new clone was identified in a hematopoietic stem cell transplanted patient. Then, 19 carbapenem-resistant P. aeruginosa were identified in an outbreak that occurred in the bone marrow transplant unit, all positive for SPM-1 gene, and 9 (47.3%) harbored both SPM-1 and KPC.All isolates were carbapenem-resistant, their MICP. aeruginosa and that this agent can harbor more than one carbapenemase gene. Attention should be focused on the possible rapid spread of KPC in P. aeruginosa isolates and for the fact that P. aeruginosa may become a reservoir of this transmissible resistance mechanism.Our findings showed that PCR for KPC gene should be performed to evaluate carbapenem resistance in  P. aeruginosa has become an important problem all over the world challenging the current diagnostic approaches. Carbapenemase genes are one of the most frequent mechanisms reported in carbapenem-resistant P. aeruginosa . Pseudomonas aeruginosa isolate co-harbouring KPC and a metallo-β-lactamase (IMP-8) was recently reported in Puerto Rico [P. aeruginosa co-harbouring SPM-1 and KPC-2 that caused an outbreak in a Bone Marrow transplantation unit, and an isolate co-harbouring three carbapenemase  that belonged to a different clone then previous described in two outbreaks that occurred in this unit and were controlled with reinforcement of hand hygiene and contact precautions, one due to P. aeruginosa harbouring SPM-1 and other harbouring VIM-2 [rature 1;. Pseudomng VIM-2 .P. aeruginosa isolate harbouring KPC-2 was identified for the first time in our hospital in 2011 in a hematopoietic stem cell transplanted patient. It harboured three carbapanemase genes , and belonged to a new clone (JA) not identified before in the hospital. This isolate showed a resistant profile to both imipenem and meropenem with MIC of 64 μg/mL and 32 μg /mL, respectively, but was susceptible to polymyxin and colistin with a MIC of 2 μg /mL for both drugs.This P. aeruginosa were identified , all positive for SPM-1 gene, and 10 (47.3%) harbored both SPM-1 and KPC-2. The majority of the KPC, six isolates of ten, belong to the clone of the outbreak (KB). Our data showed different clones circulating in our hospital and a new one predominate clone harboring KPC. A recent study also described dissemination of a new clone of P. aeruginosa harbouring KPC in a hospital in Argentina after a K. pneumonia-KPC positive outbreak [In 2012 during an outbreak that occurred in the Bone Marrow Transplant unit, 19 carbapemem-resistant outbreak .Other interesting findings of our study are that GES-5 was restricted to the Burned Intensive Care Unit and was identified only in 2001. VIM-1 was first identified in the Emergency Room and then in the Bone Marrow Transplant Unit, and SPM is spread in different units in the hospital. These results were similar with previous Brazilian reports that showed that SPM-1 is endemic in several hospitals in the country ,20.P. aeruginosa showed that they differing in size and in incompatibility group, and harbouring different genetic structures containing the blaKPC-2 genes [P. aeruginosa will be similar to that seen in Enterobacteriaceae. Thus, it is very important to understand the epidemiology of these multiresistant isolates, in order to achieve early implementation of adequate control measures to contain and reduce their dissemination in the hospital setting. Pseudomonas aeruginosa can acquired this transmissible resistance mechanism, going unnoticed and be a source of spread of KPC to other genus and species of bacteria. Besides carbapenem-resistance in P. aeruginosa can be due to two or more carbapenemase genes, including KPC-gene.The complete sequence of two KPC-harbouring plasmids, Plasmid pCOL-1 (31 529 bp), IncP-6 replicon group and Plasmid pPA-2 (7995 bp) from -2 genes . These fP. aeruginosa and this agent can harbor more than one carbapenemase gene. Attention should be focused on the possible rapid spread of KPC in P. aeruginosa isolates and for the fact that P. aeruginosa may become a reservoir of this transmissible resistance mechanism.In conclusion, our findings showed that SPM-1 is the most frequent carbapenemase identified in our hospital, followed by KPC-2. Thus, PCR for KPC gene should be performed to evaluate carbapenem resistance in


Background. Approximately one-fifth of all esophageal cancer cases are defined as early esophageal cancer (EEC). Although endoscopic therapy (ET) has been shown to be equally effective as esophagectomy (EST) in patients with EEC, there is little information comparing the survival outcomes of the two therapies based on anatomical location. Methods. A population-based study was conducted and the data was obtained from Surveillance, Epidemiology, and End Results program. Patients with EEC  and treated with either ET or EST were analyzed to compare EEC-related survival for three different locations of tumor. Results. The overall EEC-specific 1-year and 5-year mean (±SE) survival rates were 11.66 ± 0.05 and 52.80 ± 0.58 months, respectively. Tumors located in lower third had better 5-year survival compared to those located in middle third . However, when adjusted for age, race, gender, marital status, grade, stage of tumor, histological type, and treatment modality, there was no significant difference. Conclusion. The EEC-specific 1-year or 5-year adjusted survival did not differ by anatomic location of the tumor. Therefore, ET might serve as a minimally invasive yet effective alternative to EST to treat EEC. Esophageal cancer is one of the most aggressive cancers in the world. It is the eighth most common cause of cancer globally, with an estimated 456,000 newly diagnosed cases in 2012 , and the sixth leading cause of cancer death with an estimated 400,000 deaths . It is more common in developing countries in Asia and Africa, with lower incidence in Europe and North America  with microscopically confirmed stage Tis (20.3%) and stage T1a (79.8%) between 1998 and 2012 were identified. The largest proportion of tumors was located in lower third (82.7%), followed by middle third (13.2%) and upper third (4.1%) of the esophagus. The adenocarcinomas are the most common esophageal cancers constituting about 48%, 50%, and 81% of upper, mid, and lower esophagus, respectively. About two-thirds (66.8%) of the patients underwent EST, while the remaining (33.2%) had ET. p < 0.01). Tumors with stage T1a had significantly lower EEC-specific 5-year survival compared with those with stage Tis. Histologically, patients with adenocarcinoma were found to have better 1-year  and 5-year  survival compared to those with squamous cell carcinoma. EEC-specific 1-year and 5-year survival were not different between ET and EST.Overall, estimated EEC-specific 1-year and 5-year mean (±SE) survival were 11.66 ± 0.05 and 52.80 ± 0.58 months, respectively. Tables There was no significant difference in survival between ET and EST at both 1 year and 5 years for tumors localized to upper third of the esophagus. p > 0.05). Patients with “undifferentiated or grade IV” tumors had 4.12 times higher chance of dying at 5 years as compared to those with “well-differentiated or grade I” tumors .The results of the multivariate Cox proportion hazard regression analyses in In this population-based study, we found that there is no EEC-specific 1-year survival difference with respect to location of the tumor. There appears to be EEC-specific 5-year survival benefits for tumors located in the lower third compared to middle third of the esophagus. However, when adjusted for age, race, sex, marital status, size, grade, stage of tumor, histological type, and treatment modality, there was no significant difference. In addition, the treatment modality (ET or EST) did not affect 1-year or 5-year survival with respect to location of the tumor.Esophagectomy has served as the standard treatment for EEC patients due to high tumor-free survival rates. However, this procedure is associated with a mortality rate of about 1-2% at high volume centers and 5–10% at low volume centers . This prPrevious studies have compared survival outcomes between EST and ET for the treatment of EEC –9. Wani We did not observe any 1-year or 5-year EEC-specific survival benefits with respect to location of the tumor. A few studies suggest that tumor location within the esophagus impacts long-term survival , 11. In One of the advantages of the SEER database is that it is a large population-based database that includes information from multiple academic and community based institutions from around the country. This provides greater generalizability of results and allows for specific subgroups of cancers to be compared. The information collected by this database undergoes rigorous data collection procedures and quality control standards, which ensures highly accurate and reliable data.There are some limitations to our study. The SEER database does not report data on local recurrence. However, the comparable survival rates indicate that this lack of information on incomplete resection and local recurrence should not influence our overall conclusions. SEER only reports first treatment interventions in the patients. Hence recurrence of esophageal cancer and subsequent treatments were not known. Another limitation of this study is that information on comorbidities and socioeconomic factors such as smoking and alcohol use was not collected in the SEER database. It is possible that patients with more significant comorbidities were selected for ET, which could in turn have led to a decrease in magnitude of favorable outcomes with ET. Our study tried to limit this bias by focusing on EEC-specific survival rather than crude survival statistics. Also, the exact etiology of the deaths, procedure (ET and EST) related complications, and recurrence rates were not reported in SEER database.In conclusion, the results from this population-based study demonstrate that EEC-specific adjusted 1-year or 5-year survival did not differ by location of the tumors. Moreover, EEC patients treated with ET or EST exhibit comparable 1-year and 5-year survival with respect to anatomical location of tumor. Therefore, ET might serve as a minimally invasive yet effective alternative to EST to treat EEC. Further prospective randomized clinical trials are needed to validate these findings.


C) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains , middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliG Bacterial motility and chemotaxis have been studied extensively for the past few decades. These studies have established two fundamental tenets: 1. the rotation of flagellar motors is energized by membrane ion potentials  to flageSalmonella enterica serovar Typhimurium  basal bodies has been determined by electron microscopy evol by the mask matrices. The CCW mutation network had one primary node, while the CW network had several. These nodes, with two exceptions (nodes 1 and 4), mapped within or close to conserved surface residue patches . High-scoring correlations were mapped on the structures. A more stringent +3σ threshold (Methods) was used for the single correlations, relative to the +2σ threshold employed for the 6 residue nodes in the centrality profiles, with σ in both cases determined from the randomized libraries. Many correlations were between pairs greater than 20 angstroms apart in the FliMM subunit. The residues localized at subunit surfaces marked by the CW mutations, linking positions in CW nodes 1 (H1), 3  and 5 (between β1* and H2*). In-situ cross-link data have shown that these surface elements participate in inter-subunit contacts. The long-range (> 20 angstrom) correlations had comparable values to the contact (< 12 angstrom) correlations. The consequence was that correlation strength had a weak dependence on distance . The mean value / fraction above threshold for the contact (< 12 angstrom) population is 1.74 ± 0.72 / 0.15, versus 1.66 ± 0.54 / 0.1 for the non-contact (>12 angstrom) population. The dependence was insensitive to whether FliMM was in isolation, or in complex with FliGMC; though values were inflated for FliMM correlations in the complex due to inclusion of the low-scoring FliGMC correlations in the normalization. This result implies that the inter-subunit contacts are as important as the intra-subunit contacts that maintain the domain fold.We recorded the CM coevolution network results from multiple contacts between residue positions due to conformational variability between species that smear out correlations over the coevolution matrix. Superposition of the structures from the evolutionary distant T. maritima and H. pylori species does not support this explanation. The structures have a common fold , even though there are some differences [MFliGM contact, as well as FliMM fold. In contrast to FliMM self-association where residue correlations span the complete inter-subunit contact interface, the coevolution of the FliGMFliMM contact is clustered around the conserved FliMM GXGG and FliGM EHPQ motifs (see ).An alternative explanation to inter-subunit contacts is that the high-density of the FliMferences . The cortifs see  as shownM inter-subunit contacts versus the FliMMFliGM contact could evaluate the dominance of these pathways for chemotactic signal transmission. As noted, the collective FliMM W is determined by intra-domain, rather than FliGMC interactions in the composite network . The parameters used to compute these metrics from Eqs Table 2. The calculations confirm the greater strength, SM, and connectivity, C, of FliMM within the composite network. The C between FliMM residue positions is within 5% of that obtained for the randomized networks and exceeds the C of the composite network two-fold. The SM and C of the FliMM correlations with FliGMC are three and two-fold lower respectively than for the FliMM network. Interestingly, while correlations within the FliMMFliGMC contact have increased SM relative to the overall correlations between FliMM and FliGMC as might be expected for contact pairs, C is two-fold lower. The latter result shows that the contact is localized, consistent with the contact map  with elements “I, i+1, i+2 … n” of mutated residue positions, were used to create a population of dummy CW or CCW networks to estimate significance. The SM and C of the CW dummy networks generated from the lists were 0.90±0.08 and 0.91±0.05 respectively of the real CW FliMM sub-network. Thus, the CW mutations target the more prominent features of the FliMM network. This is not the case for the CCW mutations. The SM and C of the CCW dummy list was 1.04±0.17 and 1.00±0.10 respectively of the real CCW FliMM network.The CW and CCW chemotactic networks have greater (10–20%) S derived . C is alM and localized coevolution of the FliMM contact with FliGM. The FliGM EHPQ motif was a primary node in a two-point FliMMFliGM contact  sequence. The three 3HJL.pdb domains and intervening linkers form 20 α-helix segments. We focus attention on four sub-domains, N1-4 (H 1–4), ARM-M (H 7–10) ARM-C (H 12–14) and C3-6 (H 17–20) whose sequence locations are shown together with the FliG centrality in Fig 6A. The mean W values are 0.5±0.13 (N1-4), 0.4±0.1 (C3-6), 0.33±0.14 (ARM-M) and 0.18±0.1 (ARM-C). The α-helical structure of the protein is encoded in the coevolution matrix, as revealed by peaks due to the axial 3.5 residue repeat in the auto-correlation of the centrality. The peaks are absent in the auto-correlation of the randomized MSA library. The difference centrality, corrected for residual correlations, was obtained from N collectively has comparable weight, W, to FliGMC. Second, primary nodes can be identified in the difference profile. The EHPQ motif forms the dominant node 2 with the highest W, out of the seven nodes identified.The FliG coevolution matrix was generated from the MSA derived from concatenation of the Pfam FliG domain MSAs and trimmed to the full-length MFliGMC, we used the available structures to determine the dependence of correlation strength on the physical distance between correlated residues. However, conformational heterogeneity was evident in the FliGMC structures . Superposition shows that the heterogeneity as assessed by the root mean square deviation (RMSD) is due to the inter-domain linkers, since the individual domain RMSDs are lower than the overall RMSD. Within the sub-domains, ARM-C is the most, and the C3-6 the least, heterogeneous. Short-range correlations that represent contact interactions (< 12 angstrom distance (shaded block)) were notably stronger than long-range non-contact (< 12 angstrom) correlations, as shown for the two extreme conformations  . Contact correlations have 30% greater strength and over two-fold greater fraction of high-scoring correlations, F , than non-contact values. The mean strength / F for the 3AJC.pdb contact population were 2.11 ± 1.15 / 0.13, versus 1.6 ± 0.52 / 0.05 for the non-contact population. The mean strength / F for the 1LKV.pdb contact population were 2.09 ± 1.13 / 0.13, versus 1.48 ± 0.52 / 0.06 for the non-contact population.As for FliMM network in that it has distinct maxima in the centrality profile and contact distance dependence. The FliG domains have comparable W, in the network, in contrast to FliMM and FlIGMC in the composite network.In conclusion, the FliG network is different from the FliM. The images  in contact with the surrounding helices forms the core of the fold. H1 is the central helix for N1-4  architecture of the domains. The coevolution matrices for the C3-6 and ARM-M sub-domains are shown together with their centrality profiles . The nodes for the FliGMFliGC interaction network superimposed with the complete network primary nodes 2, 3 4, with a weaker contribution from primary node 7. The nodes were localized at or close to the surface. E. coli cross-link data document the surface proximity of nodes 3 and 7 through formation of FliG oligomers . Among the short-range, intra-domain networks, that for C3-6 has both the greatest SM and C; notably greater than the corresponding metrics for N1-4. The normalized SM value for C3-6 is comparable to FliMM, though C is lower. The ARM-C connectivity, C , is markedly worse than for the other modules.Computation of the SM values that are lower than for the intra-domain networks, being only marginally greater than the mean SM for the randomized networks. The C values are two-fold lower than those for the intra-domain networks. These SM differences are consistent with the stronger correlations seen between contact pairs  has somewhat higher SM than the overall FliGMFliGC interaction network, analogous to the FliMMFliGM contact. However, its correlations are uniformly distributed over the contact helices  CW network too has improved strength and connectivity  for the long-range networks to evaluate whether these formed the major determinants for the inter-domain interactions. This is the case. The FliG greater , Fig 8B.ectivity . The 3-nM domain . The C vM domain ) is clos(M (0.95 ). The himobility  as consiH. pylori FliGMC structures [C C1-6 responds as a unit to conformational changes within FliMM triggered by CheY. These changes must be relayed, in part, via the EHPQ ARM-M hub (node 2).In summary, the covariance analysis identifies a pathway for signal transmission from the EHPQ motif to the torque helix. The pathway is built from a patchwork of inter-connected nodes . Node 4 contains the MFXF motif that dominates the sparsely connected ARM-C network module. The sparse ARM-C connectivity suggests that conformational heterogeneity, seen in the superimposed X-ray structures  smears oructures , that FlC domain to, first, learn more about its evolution . The FliGC phylogenetic tree was colour coded to assess clustering. While both monophyletic and paraphyletic branches were observed, the former were predominant. The firmicutes were the most, and the δ-proteobacteria the least, monophyletic. The α-proteobacteria were the most paraphyletic; consistent with their diversity. The monophyletic branching was consistent with the neutral model of molecular evolution that posits that neutral mutations due to genetic drift are retained with selection based on phenotype while deleterious ones are rapidly eliminated . In other cases a phylum (eg. α-proteobacteria) was partitioned between disconnected branches with representatives (eg. R. Sphaeroides) divided accordingly.We constructed the phylogenetic tree of the FliGminated . For species with multiple FliG orthologues, the single FliM sequence was paired with each FliG sequence. The FliGC tree was the most compact in terms of branch length, consistent with C3-6 residue coevolution . Domain phylogenetic tree topologies were compared in duplicate for each of two reference trees (FliGM and FliGC) to check for self-consistency. Coevolution between FliGC and FliGM was detected regardless of choice of reference tree, while coevolution of these domains with either FliMM or FliGN was not. The sensitivity of similarity measures scales with sequence length and is possibly compromised by the short domain sequences. In any case, similarity detection between the FliGC and FliGM trees supported the evidence from the covariance analysis that the interaction between FliGC and FliGM was the strongest.Second, phylogenetic tree similarity offered an independent alternative, with metrics limited by different factors, to check that SM alone, FliG alone and FliMMFliGMC in complex. We separated intra-domain from inter-domain correlations, identified inter-subunit associations, and assessed network disruption by chemotactic lesions. We developed metrics based on network analysis to measure the correlations. We cannot presently relate the metrics to biochemical parameters such as binding affinity because the coevolution signal may be modulated by a number of factors as illustrated in Fig 10A. PSICOV and related algorithms have been optimized to detect hard-wired, native contacts based on static electrostatic or steric constraints, but a large macromolecular assembly such as the switch complex is likely to form a conformational ensemble with diverse dynamics. However, guided by the structural data, we are able to provide a description of the flagellar switch architecture that reveals both common elements as well as possible sources of mechanistic and species diversity.We have determined residue coevolution for FliMM indicates the importance of the FliMM fold as well as self-association. We take a high mean correlation strength, SM, and connectivity, C, of short-range contact correlations as indicators, most simply, of a compact structural fold that is conserved over species. Our data are consistent with molecular dynamics simulations that reveal the high mechanical stability of α/β/α sandwiches [M in triggering switching of rotation sense [M inter-subunit contacts suggested by the long-range correlations are consistent with this requirement and, furthermore, identify FliMM as the key determinant for the proposed conformational spread.The extended network connectivity of FliMndwiches . They aron sense ,28. Montche mutations [Salmonella CCW rotation state, as they are disrupted to a greater extent by CW mutations. Three of the four nodes in the CW mutation coevolution network map to segments previously implicated in FliM self-association. The role of the fourth node is presently unknown. The interfacial surface covered by the coevolved contacts is large. So switching would be attenuated, but not determined by the variations in subunit stoichiometry or localization of the CheY binding sites.The contacts are known targets for utations . They seC domain (C3-6) based on its coevolved network as measured by all three metrics , also has a compact fold. The torque helix H19 is central to the C3-6 coevolution network. The H10 contact correlations modify the pin-wheel architecture found for the other FliG ARM domains. This knowledge supplements the conservation of its charged residues responsible for designation of H19 as a torque helix. For torque helix movements to be entrained to C1-6 global motions [The FliG motions , it needPrimary nodes 6 and 7 flank the torque helix  and inteM and FliGC interaction network are the third feature of the common switch architecture. These nodes could constitute an allosteric relay. Studies on dihydrofolate reductase as a model system have shown that inter-connected surface sites, termed “sectors”, are preferred locations for allosteric control. These sectors were hot-spots for deleterious mutations [E. coli FliG oligomers. The E. coli cross-links could document mobility, analogous to the cross-links between nodes 4 and 7 in H. pylori  is integral to FliG network architecture, the sub-domain has sparse connectivity. Multiple factors can contribute , but theia FliGM  or direcia FliGM . The coeia FliGM  seen in evidence ,23,28. Ts argued  to accouM and FliGM. The contact is built from two FliMM residue segments in the loop at the pseudo-symmetry centre of the domain in both the T. maritima and H.pylori, structures [fliG genes whose products must both associate with a single FliM. Furthermore, FliM subunits may contact FliGC as well as FliGM within the C ring, as proposed for the E. coli flagellar motor [H. pylori for example. Strong contact between FliM and FliG is not required if the FliMM inter-subunit contacts are conserved in the common switch design to ensure conformational spread. FliG subunits can then be mobilized by the cooperative transition along the FliMM array to report FliMM conformational state to the proximal FliG C3-6 motor domain.A second element that may contribute to diversity is the contact between FliMructures  A two-poructures , as wellar motor ,49. FinaFig 10B. FliMM and FliG C3-6 form the dedicated switch and motor domains respectively of the switch complex. FliMM self-association is important for its function during chemotaxis, consistent with the proposed role of conformational spread [Our conclusions are summarized in l spread . The FliThe Methods sections correspond to the boxes in N (PF14842), FliGM (PF14841) and FliGC (PF01706) domains, and FliMM (PF02154) were downloaded from Pfam [http://www.uniprot.org) headers for concatenation of the unaligned and aligned sequences. MSA quality was assessed by measurement of the Shannon entropy of residue positions (Si).pij is the fraction of sequences at residue position i occupied by amino acid j. The entropy tends to a minimum value as conservation increases. Gaps are treated as another residue. The domain MSAs were downloaded (Pfam) or generated (CONSURF), then concatenated to obtain overall alignments. CONSURF computes residue conservation based on physico-chemical similarity [Sequences and alignments for the FliGrom Pfam . The fulrom Pfam  that wasrom Pfam . The Pfamilarity  or evoluiMM PF021 were dowi,j) in a MSA is the difference between the sum of the Shannon entropy of the individual positions  and their joint entropy, Sij. The correlation measure is the direct information, Dij, between two residue positions,Wij, Wii and Wjj are the inverse of the nMI matrices respectively [Dij values is normalized by subtraction of the mean values in the two columns for the residue positions. The coevolution matrix is formed from the normalized Dij values. Shuffling eliminates correlations between residue positions. The comparison of the real correlation value with the distribution of values from a shuffled population provided a statistical estimate of its significance. Significant correlations (“high-scoring” correlations) were taken as those whose Dij values exceeded the distribution mean by 3σ, where σ was the standard deviation of the randomized library distribution.We used the PSICOV  algorithm  to compuectively . The dishttp://www.igraph.org). Their network representations were examined with Cytoscape [The PSICOV coevolution matrices were used to generate a network model, with the residues as nodes and correlations represented by edges. Bio3D  was usedytoscape . The priC seed sequence MSA (80 sequences) to headers in the full-sequence FliGN, FliGM and FliMM MSAs. Approximately maximum-likelihood phylogenetic trees for constructed from the FliGC MSA and each of the paired MSA using Fast Tree [C tree. The process was repeated with another tree as reference. The reliability of tree splits was determined from 100 bootstrap replicates. The results were analysed by CONSEL [Domain coevolution was assessed by phylogenetic tree similarity . We pairast Tree . The paiy CONSEL . CONSEL y CONSEL  gave simy CONSEL .MFliGMC complex (4FHR.pdb), there were 2 structures of FliMM , one structure of FliGC (1QC7.pdb), 2 structures of FliMMFliGM , and 4 structures of FliGMC . These structures were of the T. maritima  or the H. pylori  proteins. The full length A. aeolicus FliG (3HJL.pdb) structure completed the set. The MSAs were processed to map residue correlations onto structure. For each structure, the associated sequence was added to the Pfam MSA with mafft-add . Residue positions absent from, or not resolved in, the structure sequence were eliminated with a custom script. The PSICOV algorithm was modified to output residue type together with residue position. The match for residue type ensured the high-scoring correlations were mapped correctly onto structure. Physical distances between correlated residue positions were computed from the Cα atoms coordinates in the maps. The Cα backbones of domains and complexes in the structures were superimposed to assess conformational heterogeneity with analysis tools in GROMACS version 4.5.5 [http://www.ks.uiuc.edu/Research/vmd) and Pymol (http://www.pymol.org/).Structures were downloaded from Protein Data Bank. In addition to the FliMon 4.5.5 . Superpoon 4.5.5  to detecS1 Fig(PDF)Click here for additional data file.